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Ku-0063794 Is a Specific Inhibitor of the Mammalian Target of Rapamycin (Mtor)

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Ku-0063794 Is a Specific Inhibitor of the Mammalian Target of Rapamycin (Mtor) Biochem. J. (2009) 421, 29–42 (Printed in Great Britain) doi:10.1042/BJ20090489 29 Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR) Juan M. GARC´IA-MART´INEZ*, Jennifer MORAN†, Rosemary G. CLARKE‡, Alex GRAY§, Sabina C. COSULICH, Christine M. CHRESTA and Dario R. ALESSI*1 *MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K., †Division of Signal Transduction Therapy, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K., ‡Division of Cell Biology and Immunology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K., §Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K., and Cancer Bioscience, Astrazeneca, Alderley Park, Cheshire, SK10 4TG, U.K. mTOR (mammalian target of rapamycin) stimulates cell growth of Thr308 and/or induces a conformational change that protects by phosphorylating and promoting activation of AGC (protein Thr308 from dephosphorylation. In contrast, Ku-0063794 does kinase A/protein kinase G/protein kinase C) family kinases such not affect Thr308 phosphorylation in fibroblasts lacking essential as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK mTORC2 subunits, suggesting that signalling processes have (serum and glucocorticoid protein kinase). mTORC1 (mTOR adapted to enable Thr308 phosphorylation to occur in the absence complex-1) phosphorylates the hydrophobic motif of S6K, of Ser473 phosphorylation. We found that Ku-0063794 induced whereas mTORC2 phosphorylates the hydrophobic motif of Akt a much greater dephosphorylation of the mTORC1 substrate and SGK. In the present paper we describe the small molecule 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than Ku-0063794, which inhibits both mTORC1 and mTORC2 with rapamycin, even in mTORC2-deficient cells, suggesting a form an IC50 of ∼10 nM, but does not suppress the activity of 76 other of mTOR distinct from mTORC1, or mTORC2 phosphorylates protein kinases or seven lipid kinases, including Class 4E-BP1. Ku-0063794 also suppressed cell growth and induced 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher aG1-cell-cycle arrest. Our results indicate that Ku-0063794 will concentrations. Ku-0063794 is cell permeant, suppresses be useful in delineating the physiological roles of mTOR and activation and hydrophobic motif phosphorylation of Akt, S6K may have utility in treatment of cancers in which this pathway is and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not inappropriately activated. regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3- Key words: Akt/protein kinase B (PKB), cancer, kinase inhibitor, phosphoinositide-dependent protein kinase-1). We interpret this phosphoinositide 3-kinase (PI3K), p70 ribosomal S6 kinase as implying phosphorylation of Ser473 promotes phosphorylation (S6K), serum and glucocorticoid protein kinase (SGK). INTRODUCTION activated protein kinase) pathway [11]. mTORC2 consists of mTOR, Rictor, Sin1, mLST8 as well as Protor and is insensitive The mTOR (mammalian target of rapamycin) protein kinase to acute rapamycin treatment [12–18]. The activity of mTORC2 lies at the nexus of signalling pathways that regulate cell activity is controlled by PI3K, but is insensitive to amino acids or growth and proliferation. Many cancer-driving mutations in genes energy stress. such as receptor tyrosine kinases, Ras, PI3K (phosphoinositide A key role of mTOR complexes is to phosphorylate and 3-kinase) and PTEN (phosphatase and tensin homologue deleted control the activity of a subgroup of related AGC (protein kinase on chromosome 10) stimulate proliferation, growth and survival A/protein kinase G/protein kinase C) family kinases that comprise by activating mTOR kinase. Two mTOR complexes have isoforms of Akt, S6K (p70 ribosomal S6 kinase), PKC (protein been characterized, termed mTORC1 (mTOR complex-1) and kinase C) and SGK (serum and glucocorticoid protein kinase). mTORC2 (reviewed in [1,2]). mTORC1 consists of mTOR, mTOR complexes phosphorylate these enzymes at a conserved Raptor and mLST8 and is acutely inhibited by rapamycin [3–5]. C-terminal non-catalytic residue, termed the ‘hydrophobic motif’. mTORC1 is activated by insulin and growth factors via a PI3K- Activation of AGC kinases also requires phosphorylation of controlled pathway, involving Akt-mediated phosphorylation of an additional site within their kinase catalytic domain termed PRAS40 (proline-rich Akt substrate of 40 kDa) and tuberous the ‘T-loop motif’. This phosphorylation is executed by the sclerosis complex-2 protein, and activation of the Rheb GTPase PDK1 (3-phosphoinositide-dependent protein kinase-1) [19]. [6,7]. mTORC1 is also stimulated by amino acids through a mTORC1 phosphorylates the hydrophobic motif of S6K [4,5], pathway involving bidirectional transport of amino acids [8] and whereas mTORC2 phosphorylates the hydrophobic motifs of Akt the Rag GTPase [9,10]. Low-energy conditions suppress cell [20], SGK [21] and certain isoforms of PKC [17,22]. In the growth by inhibiting mTORC1 via the LKB1–AMPK (AMP- case of S6K, SGK and PKC isoforms, hydrophobic motif Abbreviations used: AGC family, protein kinase A/protein kinase G/protein kinase C family; AMPK, AMP-activated protein kinase; DTT, dithiothreitol; eIF4E, eukaryotic initiation factor 4E; 4E-BP1, eIF4E-binding protein 1; ERK, extracellular-signal-regulated kinase; GST, glutathione transferase; HEK-293 cell, human embryonic kidney 293 cell; IGF, insulin-like growth factor; MAPK, mitogen-activated protein kinase; MEF, mouse embryonic fibroblast; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; NDRG1, N-Myc downstream-regulated gene-1; PDK, 3-phosphoinositide-dependent protein kinase; PH domain, pleckstrin homology domain; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PRAS40, proline-rich Akt substrate of 40 kDa; RSK, ribosomal S6 kinase; S6K, p70 ribosomal S6 kinase; SGK, serum and glucocorticoid protein kinase; SPHK, sphingosine kinase;. 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2009 Biochemical Society 30 J. M. Garc´ıa-Mart´ınez and others phosphorylation creates a high-affinity docking site for PDK1, termed pNDRG1 3 × Thr-P) was raised against the nonapeptide thereby promoting phosphorylation and activation of these RSRSHpTSEG, whose sequence is common to all three SGK1 enzymes by PDK1 [19,23]. In the case Akt, interaction of phosphorylation sites on NDGR1 (used for immunoblotting). the PH (pleckstrin homology) domains in PDK1 and Akt with Anti-Akt1 (S695B, 3rd bleed; residues 466 – 480 of human Akt1 PtdIns(3,4,5)P3 (a product of PI3K activation), at the plasma RPHFPQFSYSASGTA, used for immunoblotting and immun- membrane of cells, brings these enzymes together and induces a oprecipitation), anti-S6K (S417B, 2nd bleed; residues 25–44 of conformational change in Akt that enables it to be phosphorylated human S6K, AGVFDIDLDQPEDAGSEDEL, used for immun- at Thr308 by PDK1 [24,25]. Recent data also indicates that oblotting and immunoprecipitation). Anti-GST (S902A, 1st bleed) mTORC2 controls the phosphorylation of Akt and PKC isoforms was raised against the GST tag expressed from pGex4T (used for at a conserved phosphorylation site termed the ‘turn motif’ immunoblotting). Anti-PRAS40 (S115B, 1st bleed; residues 238– that stabilizes these enzymes in an active conformation [26,27]. 256 of human PRAS40, DLPRPRLNTSDFQKLKRKY, used mTORC1 also phosphorylates other substrates such as the for immunoblotting). An antibody that recognizes PRAS40 eukaryotic translation initiation factor 4E-BP1 [eIF4E (eukaryotic phosphorylated at Thr246 (S114B, 2nd bleed) was raised against initiation factor 4E)-binding protein 1]. Phosphorylation of 4E- the residues 240–251 of human PRAS40, CRPRLNTpSDFQK BP1 by mTOR induces dissociation from eIF4E, promoting (used for immunoblotting). Anti-RSK2 (ribosomal S6 kinase 2) initiation of protein translation that is required to power cell (S382B, 1st bleed; residues 712–734 of human RNQSPVLEPV- growth and proliferation ([4,5],[28], but see [28a]). In the present GRSTLAQRRGIKK, used for immunoblotting and immuno- study, we describe the characterization of Ku-0063794, a novel precipitation); Anti-FoxO1 (S504A, 1st bleed) was made in sheep potent and highly specific small-molecule inhibitor of the mTOR using recombinant GST-fusion FoxO1 (2–655, used for im- protein kinase. We demonstrate that Ku-0063794 inhibits both munoblotting). Anti-β-tubulin (H-235, #sc-9104), phospho-RSK mTORC1 and mTORC2 in vitro and in vivo and can be employed Thr227 (#sc-12445-R) and the total mTOR antibody (#sc-1549) to dissect cellular functions of the mTOR pathway. were purchased from Santa Cruz Biotechnology. For phospho- immunoblotting of the hydrophobic motif of SGK1(Ser422), we 389 MATERIALS AND METHODS employed the Thr S6K antibody (# 9205) from Cell Signaling Technology, which we previously demonstrated cross-reacted Materials with the phosphorylated Ser422 of SGK1 [30]. The phospho- 473 308 450 Protein G–Sepharose and glutathione–Sepharose were purchased Akt Ser (#9271), Thr (#4056), Thr (#9267), phospho-S6K 389 235 from Amersham Bioscience. [γ -32P]ATP was from PerkinElmer; Thr (#9234), phospho S6 ribosomal protein Ser (#4856), IGF1 (insulin-like growth factor) was from Invitrogen.
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